1. Technical Field
The subject invention relates to the construction of antisense oligonucleotides of the human Chk1 gene and to uses thereof. The human Chk1 gene is a major G2/M checkpoint gene that is activated in response to DNA damage. In particular, the gene transduces the inhibitory signal from DNA damage sensors to the basic cell cycle machinery. Thus, antisense oligonucleotides to the human Chk1 gene may be used to inhibit gene expression thereby preventing G2 arrest induced by DNA damaging agents. Additionally, antisense oligonucleotides may act to sensitize tumor cells thereby making them more sensitive to therapy than normal cells.
2. Background Information
Many cancer therapeutic reagents cause cell death by inducing severe cellular DNA damage. Such DNA damage elicits two responses in normal, eukaryotic cells: 1) cell cycle arrest and 2) DNA repair to maintain genetic fidelity. In particular, checkpoint genes are activated in response to DNA damage. The gene products of these checkpoint genes form signal transduction pathways that transduce inhibitory signals from the DNA damage sensors to the basic cell cycle machinery and result in cell cycle arrest at both the G1 and G2 phases. Simultaneously, DNA damage also induces activation of transcription and production enzymes that facilitate DNA repair (Paulovich et al., Cell 88:315-321 (1997))).
p53 is the major G1 phase checkpoint gene. This protein is activated in the event of DNA damage (Shieh et al., Cell 91:325-34 (1997); Kastan et al., Cancer Research 51:6304-11 (1991)). (It transcriptionally activates cell cycle inhibitors such as p21, which, in turn, inhibit G1 cyclin-Cdks, thereby preventing cells from traversing the G1/S boundary (el-Deiry et al., Cancer Research 54:1169-74 (1994); Dulic et al., Cell 76:1013-1023 (1994)).
At the G2/M boundary, the onset of mitosis depends on the active cyclin B-Cdc2 kinase complex (King et al., Cell 79:563-71 (1994); Lew et al., Current Opinion in Cell Biology 8:795-804 (1996)). Weel kinase and Cdc25C phosphatase regulate Cdc2 activity. In particular, Weel phosphorylates Cdc2 at tyrosine 15 which inactivates Cdc2. Cdc25C removes this inhibitory phosphate and keeps Cdc2 active (Nurse et al., Cell 91: 865-7 (1997)).
During the G2 phase, DNA damages leads to Chk1 phosphorylation and activation in an ATM dependent manner (Walworth et al., Science 271:353-6 (1996)). Active Chk1 phosphorylates Cdc25C at serine 216, and 14-3-3 proteins export the phosphorylated Cdc25c out of the nucleus of the cell. Thus, Cdc2 is inhibited by the phosphorylation at tyrosine 15, and cells are halted at G2 for DNA repair (Furnari et al., Science 277:1495-7 (1997); Furnari et al., Molecular Biology of the Cell 10:833-45 (1999); Sanchez et al., Science 277:1497-501 (1997); Peng et al., Science 277:1501-5 (1997)).
It has been known for some time that the majority of tumors have defects in G1 checkpoint machinery, many of them due to p53 mutations (Kastan et al. (1991) Cancer Research 51:6304-11). In the event of DNA damage, these G1 checkpoint-defective tumor cells depend primarily on the G2 checkpoint for DNA repair. The inability to repair DNA at the G1 checkpoint is consistent with the observation that tumor cells are more sensitive to DNA damaging therapeutics than are normal cells. Given this observation, the toxicity to normal tissues is still a common side effect in cancer therapy. Significant effort has therefore been expended to specifically sensitize tumors to cancer drugs or radiotherapy.
One such approach is to abrogate G2 arrest in response to DNA damage (Powell et al., Cancer Research 55: 1643-8 (1995); Yao et al., Nature Medicine 2:1140-3 (1996)). Since G1 checkpoint-defective tumor cells can only repair DNA in G2 phase, inhibition of the Chk1 gene is expected to a brogate the G2 arrest in DNA damage response and increase the sensitivity to chemotherapy/radiotherapy more profoundly in tumor cells than in normal cells.
All U.S. patents and publications are herein incorporated in their entirety by reference.